Attachment of blastocysts to lens capsule: A model system for trophoblast-epithelial cell interaction on a natural basement membrane

Summary The bovine lens capsule has previously been shown to provide an optimal surface for the examination of epithelial cell interaction with a basement membrane. This native substrate has been used to investigate some initial aspects of attachment of mouse blastocysts and trophoblastic cellular outgrowth. Mouse blastocysts were presented to the cell-free humoral side of the anterior lens capsule, incubated for 72 h, and examined by scanning and transmission electron microscopy. Blastocysts hatch and attach from their zonae pellucidae by 30 h. Trophoblastic cells proliferate rapidly in a coronal direction, display extensive surface microvilli, and advance by the extension of numerous filipodia, many of which terminate with bulbous projections. These projections were shown by transmission electron microscopy to contain numerous vacuoles and polysomes. To simulate further the initial blastocyst-uterine interaction, a suspension of lens epithelial cells was introduced to the capsule and permitted to form a monolayer prior to the addition of the blastocysts. At 72 h the monolayer of lens cells remained intact. We observed that: a) lens cells appear to recede from the advancing trophoblastic cells, and b) trophoblastic cells extend beneath the monolayer of lens cells and thereby dislodge the cells from the lens capsule substrate. No infiltration of the capsule by the advancing trophoblastic cells was observed. The lens capsule appears to offer a promising system for the study of trophoblast-epithelial cell interaction on a natural basement membrane.     

Thermal inhibition of repair of methylmethane sulfonate-damaged DNA in chick embryo fibroblasts

Chick embryo fibroblasts were treated with the monofunctional alkylating agent methylmethane sulfonate at various concentrations for 1 h at 42°C, rinsed and then incubated post-treatment at various temperatures at which the kinetics of alkali-labile bond disappearance was followed. Growth experiments showed that these cells grew similarly at temperatures of either 37°C or 42°C. Repair as assessed by removal of alkali-labile bond was also similar for postincubation in the temperature range 37–42°C for damage due to methylmethane sulfonate treatment at concentrations less than 1.5 mM. When the postincubation temperature was raised higher than 42.5–43°C, this type of repair was stopped. The normal internal body temperature of adult chickens is about 41.6°C. Hence the present finding indicates that chick cells are much more severely restricted in DNA repair at temperatures above normal than are mammalian cells, which can function in this respect for several deg. C above 37°C.     

Metabolic plasticity drives development during mammalian embryogenesis

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Metabolic rate in diapause and nondiapause brown locust eggs correlated with embryonic development

Insects use dormancy to survive adverse conditions. Brown locust Locustana pardalina (Walk.) eggs offer a convenient model to study dormancy (diapause and quiescence), which contributes to their survival under arid conditions. The metabolic rates of developing nondiapause, diapause and quiescent eggs are compared in the present study using closed‐system respirometry. The embryo becomes committed to continue development and hatch or to enter diapause 6 days after the eggs are placed on moist soil. The metabolic rate of nondiapause eggs increases exponentially until hatching, whereas that of diapause eggs is low and stable. The metabolic rate of diapause laboratory eggs (1.9 ± 0.6 µL CO₂ mg^?1 h^?1) is significantly higher than that of field eggs (0.5 ± 0.3 µL CO₂ mg^?1 h^?1), although the ranges of metabolic rate overlap and the embryos are all in late anatrepsis. The metabolic rate of quiescent eggs is similar to that of diapause eggs but decreases with time. Low metabolic rates during arrested development allow eggs to persist over long periods before hatching.     

Embryo culture of Medicago scutellata and M. sativa

Resistance to the alfalfa weevil (Hypera postica (Gyllenhal)) and the potato leafhopper (Empoasco fabae (Harris)) is lacking in cultivated alfalfa. However, a closely related annual Medicago, Medicago scutellata, possesses dense glandular stem and leaf hairs which provides a mechanism for resistance. Several attempts have been made at transfering the glandular haired traint from M. scutellata to perennial alfalfa with limited success. Earlier studies have shown that one reason for the lack of success is embryo abortion. Therefore, this study was initiated to observe zygotic embryo-genesis and to develop an embryo rescue technique for M. scutellata and M. sativa. Observations of zygotic embryogenesis showed that the two species are similar in morphology and can be described from youngest to oldest as globular, heart, torpedo, and hook shaped embryos. M. sativa embryos are smaller than M. scutellata embryos and develop three to four days later. Self pollinated M. scutellata (PI 307446) and sib mated M. sativa (Saranac AR) embryos were cultivated on Murashige and (2,4-D), indolacetic acid (IAA), 6-benzylaminopurine (BAP), and kinetic (KIN). Embryos from both species were also cultured on Schenk and Hildebrandt's (SH) basal medium with the addition of L-glutamine and L-proline. The experimental design was a completely randomized factorial for each experiment. Heart and torpedo shaped embryos from M. scutellata grew best (27.5% plantlet recovery) when cultured on MS medium with 0.05 mgl^-1 of both IAA and BAP. After 15 to 30 days on this medium, the embryos had only developed shoots. Therefore, it was necessary to transfer the shoots to MS basal medium without phytohormones for rooting. Rooting occurred in 15 to 30 days and the plantlets could be acclimatized to soil within 2 to 4 weeks. M. sativa embryos grew best (31% plantlet recovery) on SH medium with 50 mM L-glutamine. M. sativa embryos developed both shoots and roots on this medium. This information may now be applied to the development of an embryo culture method for recovering insect resistant hybrids between M. scutellata and M. sativa. Disclaimer statement: Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the USDA, and does not imply its approval to the exclusion of other products that may also be suitable.     

Cell junctions in the rapidly moving edge of chick and quail blastoderms during gastrulation

Summary The intercellular contacts of the migrating edge of chick and quail blastoderms during gastrulation were studied by transmission electron microscopy of thin sections and of freeze-fracture replicas. Tight junctions and gap junctions as well as desmosomes were found. Tight junctions were organized as single junctional strands or as a complex of numerous junctional strands interposed between the lamellae and the bodies of the cells building up the margin of overgrowth. The function of these intercellular junctions is considered in relation to the locomotion of the margin of overgrowth cells.     

Stimulator of interferon genes (STING): A “new chapter” in virus-associated cancer research. Lessons from wild-derived mouse models of innate immunity

Thanks to the numerous studies that have been carried out recently in the field of cytosolic DNA sensing, STING (Stimulator of Interferon Genes) is now recognized as a key mediator of innate immune signaling. A substantial body of evidence derived from in vivo mouse models demonstrates that STING-regulated pathways underlie the pathogenesis of many diseases including infectious diseases and cancers. It has also become evident from these studies that STING is a promising therapeutic target for the treatment of cancer. However, mouse strains commonly used for modelling innate immune response against infections or tumors do not allow investigators to accurately reproduce certain specific characteristics of immune response observed in human cells. In this review, we will discuss recent data demonstrating that the use of wild-derived genetically distinct inbred mice as a model for investigation into the innate immunity signaling networks may provide valuable insight into the STING-regulated pathways specific for human cells. The maximum complexity of STING-mediated mechanisms can probably be seen in case of DNA virus-induced carcinogenesis in which STING may perform unexpected biological activities. Therefore, in another part of this review we will summarize emerging data on the role of STING in human DNA virus-related oncopathologies, with particular attention to HPV-associated cervical cancer, aiming to demonstrate that STING indeed “starts a new chapter” in research on this issue and that wild-derived mouse models of STING-mediated response to infections will probably be helpful in finding out molecular basis for clinical observations.     

Comparison of the relative sensitivity of human lymphocytes and mouse splenocytes to two spindle poisons

Aneugenic compounds act on non-DNA targets to exert genotoxicity via an indirect mechanism. In contrast to DNA-binding agents, these compounds are expected to possess threshold levels of activity. Therefore, the risk for adverse effects following human exposure to an aneugen could be minimal, if the threshold of activity has been clearly determined in vivo and in vitro and providing the human exposure level is below this threshold. Thus, the development of a single-cell model to allow comparisons between in vitro and in vivo threshold values for aneugenic compounds is of importance.The in vivo micronucleus test is one of the main assays used in genetic toxicology, and is often performed in the mouse. Thus, an extensive database is available in the literature. However, there are only few data concerning the in vitro micronucleus assay using mouse cells, as the majority of in vitro micronucleus assays have been performed using human lymphocytes. In addition, there is a lack of data concerning thresholds for any compound using this model.First, we evaluated whether the use of mouse splenocytes would be an acceptable alternative to that of human lymphocytes to identify aneugens. To allow valid comparisons, the two protocols were first harmonized. Thus, phytohemagglutinin (PHA) and concanavalin A were used as specific mitogens for human lymphocytes and mouse splenocytes, respectively, in order to achieve similar cell-proliferation rates. To achieve similar and sufficient numbers of binucleated cells, cytochalasin B was added 44 and 56 h after culture initiation of the human and mouse cells, respectively.Second, we compared the sensitivity of the mouse protocol with that of the human protocol by exposing the cells to the aneugens nocodazole and paclitaxel.There was good reproducibility of the cytotoxic/genotoxic responses of the two cell models following exposure to the aneugens. The sensitivity of the mouse splenocytes to paclitaxel was higher than that of the human lymphocytes. The two cell types were equally sensitive to nocodazole.